Team:Newcastle/6 August 2010
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*See [[Team:Newcastle/Ligation|Ligation]] protocol | *See [[Team:Newcastle/Ligation|Ligation]] protocol | ||
*See [[TeamNewcastleTransformation_of_E._coli|Transformation]] protocol | *See [[TeamNewcastleTransformation_of_E._coli|Transformation]] protocol | ||
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+ | ==Inference== | ||
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+ | We expect cells transformed with lacI and pVeg to survive on the ampicillin plates. On Monday we will transform ''E.coli'' with the ligation product. | ||
{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} |
Revision as of 13:25, 6 August 2010
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Contents |
Gel Electrophoresis for the Amplified Pspac_oid promoter and lacI
Aim
The aim of the experiment is to perform gel electrophoresis for the two PCR reactions viz. lacI and Pspac_oid promoter which took place yesterday 5th August, 2010 and thus confirm that they were successful.
Materials and Protocol
Please refer to: Gel electrophoresis.
Result
- Lane 1: 1kb DNA ladder
- Lane 2: Plamid pMutin4 containing lacI
- Lane 3: Plamid pMutin4 containing Pspac_oid promoter
- Lane 4: 100bp DNA ladder
Biobrick compatible vector pSB1C3 | Pspac_oid pormoter | 1st fragment of rocF CDS | 2nd fragment of rocF CDS) | 3rd fragment of rocF CDS | Double Terminator | |
---|---|---|---|---|---|---|
Size of the Fragment (in bp) | 2072 approx. | 106 approx. | 246 approx. | 597 approx. | 125 approx. | 116 approx. |
Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
Discussion
We found bands in the lanes 2,4,5,6 and 7 of the correct sizes but lane 3 did not contain any band.
Conclusion
This experiment shows that the PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was present in the lane 3 and did not show any band. This could be because of the following problems:
- Primer sequences could be incorrect.
- Melting temperature could be incorrect.
- Plasmid pMutin4 could have degenerated due to long term storage.
lacI ligation(repeat) and Transformations
Aims
In this experiment we aim to tranform competent E.coli DH5-alpha with pVeg spoVG and lacI in preparation for Gibson cloning. We also aim to repeat the ligation from week 12-17th July.
Materials and Protocols
- See Ligation protocol
- See Transformation protocol
Inference
We expect cells transformed with lacI and pVeg to survive on the ampicillin plates. On Monday we will transform E.coli with the ligation product.
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